An ideal polymer to be used as carrier for drug delivery or molecular imaging should be characterized by (i) biodegradability or adequate molecular weight that allows elimination from the body to avoid progressive accumulation in vivo; (ii) low polydispersity, to ensure an acceptable homogeneity of the final system allowing to adjust pharmacokinetics; (iii) longer body residence time either to prolong the conjugate action or to allow distribution and accumulation in the desired body compartments (therefore high molecular weights are desired); and (iv) for protein conjugation, only one reactive group to avoid crosslinking (semitelechelic polymers), whereas for small drug conjugation, many reactive groups to achieve a satisfactory drug loading (multivalent polymers).
Because of their implicit nature, polymers present specific challenges for the pharmaceutical development. A manufactured drug substance should be homogeneous and composed of single, defined specie. By contrast, all synthetic polymers are inherently heterogeneous and, as macromolecules, they can bear special challenges for characterization. The total control of crucial parameters such as molecular weight, polydispersity, localization of charge or hydrophobicity-hydrophilicity balance is a must in order to tune body biodistribution, fate, biological activity and toxicity [1,2]. The average molecular weight is described by the “weight average molecular weight” (Mw) and “number average molecular weight” (Mn) and the ratio Mw/Mn gives a measure of the dispersity D.
Therefore there is an interest in finding methodologies to enhance polymer molecular weight for biodegradable systems controlling the homogeneity in the process and allowing a high degree of versatile to be implemented in different clinical needs.
The ring-opening polymerization (ROP) of amino acid-N-carboxyanhydrides (NCA) is the most commonly applied polymerization technique to produce polypeptides and polypeptide-based block copolymers on a several gram scale. Although the obtained polymers are less defined than peptides produced by a natural organism the polymerization method enables access to polypeptidic architectures, which are beyond nature's possibilities. Furthermore the ROP of NCAs has already been applied to various applications in different fields of science. Those applications range from drug delivery systems or molecular imaging systems to surface coating materials [2-6].
As a prominent example for the use of the NCA polymerization one has to mention a conjugate of polyglutamic acid (PGA) and paclitaxel (Opaxio®, formerly Xyotax, PPX, CT-2103). The polymer drug conjugate has entered clinical phase 3 trails 7-[9] emphasizing the importance of the NCA polymerization method for the preparation of well-defined synthetic polypeptides. Polyglutamic acid is promising material for the design of nanomedicines due to its high biocompatibility, multivalency and in vivo degradability by thiol proteases (cathepsin B). [10-11]
From the historic point of view the NCA polymerization is a rather old method. It was discovered by Leuch in the beginning of the 20th century. [12-14] In respect to this fact various methods have been reported for the ROP of NCAs as reviewed in following excellent literature. [15-17] So far the most promising chemical approaches are based on initiation of purified NCAs with primary amines and high vacuum techniques, [18-20] use of amine hydrochloride salts as initiators [21], heavy metal catalysts [22-24] or hexamethyldisilazanes (HMDS) [25-26]
Unfortunately, most of those methods have certain limitations in the synthesis of well-defined polypeptides. Hexamethyldisilazanes (HMDS) amines are sensitive to hydrolytic reactions. While heavy metal catalysts have to be removed afterwards whenever biomedical applications are desired. The removal is both time consuming and incomplete.
The normal initiation with a primary amine (NA) leads in most cases to reduced control about the polymerization process itself. Especially whenever a higher degree of polymerization or complex architectures are desired the occurring side reactions interfere. In general Polyglutamates with a molecular weight average ranging from some thousands up to 50 kg/mol and PDIs of 1.2 to 1.5 are reported in literature [17]. Large PDI values are in all probability to be attributed to the fact that NCA polymerization suffers from side reactions. The most likely one is the “activated monomer” process (AM), initiated by the deprotonaton of an NCA molecule. The NCA anion is a sufficiently strong nucleophile to initiate the oligomerization of NCAs. The formed N-aminoacyl NCA compounds will either add to the propagating chain end or undergo self-condensation, the latter reaction producing high molecular weight products at high monomer conversion. Since primary amines can act as both a nucleophile and a base, polymerization will always switch back and forth between the “amine” and the “activated monomer” mechanism (NAM).
By lowering the reaction temperature the polypeptides may get more defined, because side reactions are suppressed, but reaction times increase about 2-4 times while yield decreases [27-29]. However, for the production of block copolymers (diblock, triblock or multiblock), NCA polymerization should preferably proceed until high conversions via the “amine” mechanism, i.e. nudeophilic ring-opening of the NCA leading to defined end groups. The control over polymer end groups is essential for the synthesis of multiblock architectures. [30]
The “activated monomer” pathway might be avoided simply by adding an acid, inducing the re-protonation of eventually formed NCA anions. Nevertheless this idea is not new. Basically, this idea goes back to the work of Knobler et al. published in the 1960s [31-32]. These authors investigated the stoichiometric reaction between NCAs and the hydrochlorides of primary amines for the preparation of aminoacyl compounds. Schlaad and coworkers have used this method to prepare well-defined Polystyrene based block copolymers. [21]
The disadvantage of this approach is the fact that the chloride ion itself can act either as a nucleophile or base deprotonating the NCA and therefore side reactions e.g., initiation of the NCA ring opening by the activated monomer mechanism (AMM) as already demonstrated by Schlaad [21] and was reproduced in our lab (for analytical data of the HCl induced polymerization of glutamic acid based NCAs is included for comparison (see Table 1)).
TABLE 1Comparison between Normal Amine and Schlaad NCA polymerization approaches.DPt[M]/DP1H-[M]reactionInitiatorP[I]Calc. a)NMRMol(d)YieldMnMwPDINAM110077410.3837720.325.41.22400300590.3837522.431.91.431601328600.3838320.528.31.4  Schlaad410060360.383604.45.61.35400288550.3837218.127.21.561600 1168900.3837320.731.21.5 P: Polymer,[M]/[I]: Monomer to Initiator ratio;DP: Degree of polymerization (% of [M]/[I];)Mw: weight average molecular weight;Mn: number average molecular weightPDI: polydispersity index (ratio Mw/Mn that gives a measure of the dispersity D).                                       a          )                    ⁢      Calculated        ⁢                  ⁢    using    ⁢                  ⁢    DP    =                    [        M        ]                    [        I        ]              ·    con  
As shown in Table 1, although both methods are easy to apply without complicated equipment or complex synthesis they are invalid whenever a degree of polymerization (DP) above 100 is desired. Thus, there is a need for a different approach.